The klotho gene was originally identified as a suppressor of premature aging [1, reviewed in 2]. Klotho is a single-pass transmembrane protein expressed predominantly in kidney, the parathyroid gland, and the choroid plexus [1, 3, 4]. Paralogous proteins with distinct functions and expression profiles, termed βKlotho and γKlotho [5, 6] are also known.
αKlotho has diverse effects including regulating ion transport, Wnt and insulin signaling, renin-angiotensin system, recruitment of stem cells, anti-carcinogenesis, anti-fibrosis, and antioxidation. The highest level of expression of αKlotho is in the kidney [1, 7, 8]. In addition to its transmembrane form which is a co-receptor for fibroblast growth factor (FGF) 23, [9-11] αKlotho is also released into the circulation, urine, and cerebrospinal fluid as an endocrine substance[7, 12, 13] generated by transcript splicing into a truncated peptide[2] or proteolytic release by secretases.[14, 15] A substantial portion of the circulating αKlotho is nephrogenic in origin[16]. The phenotypic similarities between genetic αKlotho ablation and chronic kidney disease (CKD) support the notion that reduced renal expression of αKlotho is pathogenic[1, 16].
Reduced renal αKlotho transcript or protein levels[12,18-24] and serum αKlotho concentration[12, 20] was demonstrated in rodent CKD from nephron reduction surgery, ischemia reperfusion injury, immune complex glomerulonephritis, polygenic or hormonal hypertension, metabolic syndrome, and diabetes.[12, 18-24] This convergence suggests that αKlotho deficiency may be a generic consequence of nephron loss. αKlotho reduction is potentially a sensitive and early biomarker of CKD and also prognostic of CKD complications [22]. Restoration of αKlotho in experimental CKD in rodents ameliorates the kidney disease and extra-renal complications [12, 22, 23]. αKlotho deficiency has also been documented in acute kidney injury (AKI) in both rodents and humans [25]. αKlotho can potentially serve as an early biomarker for AKI as it is reduced much earlier than changes in the current known biomarkers of AKI [26].
αKlotho forms a constitutive binary complex with FGF receptors (FGFRs) to confer selective affinity to FGF23 [10, 27]. Defects in αKlotho expression result in FGF23 resistance and phosphate retention in mice [1, 28] and humans [29]. Therefore, αKlotho and FGF23 have emerged as essential components of the bone-kidney endocrine axis that regulates phosphate metabolism [30, 31].
The extracellular domain of the membrane-anchored form of αKlotho can be secreted as a soluble protein. The soluble form is generated from the membrane-anchored form by membrane-anchored proteases and is released into blood and urine [13, 15]. As noted above, membrane-anchored αKlotho functions as part of the FGF23 receptor complex, whereas secreted αKlotho functions as an endocrine factor that exerts actions on distant organs to exert highly pleiotropic actions as stated above (regulating ion transport, Wnt and insulin signaling, renin-angiotensin system, recruitment of stem cells, anti-carcinogenesis, anti-fibrosis, and antioxidation) [7].
Advanced CKD (Stages 4-5), characterized by kidney damage and decreased kidney function, affects an estimated 2.6 million Canadians, greater than 7% of the population. A recent analysis of National Vital Statistics Report, National Health and Nutrition Examination Surveys and US Renal Data System showed that the lifetime risks for white men, white women, black men, and black women, respectively: CKD stage 3a+, 53.6%, 64.9%, 51.8%, and 63.6% [84]. The impact and burden of CKD and its associated complications on people's lives and the health care system is significant and will worsen in coming years [32-34]. Current approaches to treat CKD include modification of risk factors by diet and medication, and for end stage renal disease (ESRD) by dialysis, and organ replacement. There is an urgent need for additional therapies to, arrest or delay progression of CKD at early stages, before complications arise. The majority of the complications of CKD are embraced within the entity of CKD-mineral bone disturbance (CKD-MBD) which are tied to disturbances of mineral metabolism. Phosphate retention is universally observed in CKD patients and associated with poor outcome [35, 36]. Hyperphosphatemia is usually detected only in advanced stages of CKD, when the disease is destined to progress to end-stage [37].
Recently, it has been discovered that reduced renal αKlotho expression is one of the earliest events in CKD [12].
At present, there are some αKlotho antibodies and diagnostic kits available on the market, but the existing αKlotho antibodies are not of sufficient specificity and not efficient at immunoprecipitating αKlotho from human serum, and the current immune-based assays for αKlotho are costly and inadequate in sensitivity and specificity.
Low αKlotho transcript and protein levels have been described in human kidney from nephrectomy samples of end stage kidneys and biopsies from patients with CKD [21,38]. Studies using an immune-based assay have shown widely disparate results in terms of absolute values of serum αKlotho concentration (100-fold span in levels from different labs) and direction of change (increased, decreased, or no change) with CKD and age[21,39-60]. The discrepant database has thwarted progress and incapacitated the ability to determine whether the promising rodent data can be translated into meaningful human application. In addition to CKD, acute kidney injury (AKI) from a variety of causes is also associated with rapid decrease of αKlotho in the kidney[25, 61-65] and serum in rodents and in urine in humans[25]. There is no data on human serum αKlotho in AKI to date. There is a need for an early, sensitive, and/or specific marker for renal injury in humans[66].
Generating antibodies to conserved proteins is challenging, as animal immunization methods for antibody development are subject to mechanisms that protect against auto-immunity. Synthetic antibody technology offers a powerful alternative because it is applied under defined in vitro conditions, uses antibody libraries that have not been subjected to tolerance selection that remove self-reactive antibodies, and is proven to yield antibodies with high affinities and specificities[67-71]. Within an optimized antibody framework, sequence diversity is introduced into the complementary determining regions (CDR's) by combinatorial mutagenesis. These libraries are coupled with phage display, with each phage particle displaying a unique antigen-binding fragment (Fab) on its surface while carrying the encoding DNA internally, thus achieving direct phenotype-genotype relations. Fab-displaying phage that bind to an antigen of interest are enriched using binding selections with purified antigens on solid support. The CDR's of binding phage clones are identified by DNA sequencing and the Fab proteins are purified from bacteria, or converted to the full-length IgG in mammalian cells.